Tuberculosis-specific antigen stimulated and unstimulated interferon-γ for tuberculous meningitis diagnosis: A systematic review and meta-analysis

Objective Tuberculous meningitis (TBM) is one of the most devastating TB. Accurate identification of TBM is helpful to eliminate TB. Therefore, we assessed the performance of TBAg stimulated IFN-γ (IGRA) and unstimulated IFN-γ in blood and cerebrospinal fluid (CSF) for diagnosing TBM. Methods We searched Web of Science, PubMed, Embase and the Cochrane Library databases until March 2022. Bivariate and hierarchical summary receiver operating characteristic models were employed to compute summary estimates for diagnostic accuracy parameters of IGRA and unstimulated IFN-γ in blood and CSF for diagnosing TBM. Results 28 studies including 1,978 participants and 2,641 samples met the inclusion criteria. The pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR) and area under the curve (AUROC) of blood IGRA were separately as 0.73, 0.83, 4.32, 0.33, 13.22 and 0.86, indicating a good diagnostic accuracy of blood IGRA for detecting TBM. The summary sensitivity, specificity, PLR, NLR, DOR and AUROC of CSF IGRA were separately as 0.77, 0.91, 8.82, 0.25, 34.59 and 0.93, indicating good diagnostic accuracy of CSF IGRA for detecting TBM. The summary sensitivity, specificity, PLR, NLR, DOR and AUROC of CSF IFN-γ were separately as 0.86, 0.92, 10.27, 0.16, 65.26 and 0.95, suggesting CSF IFN-γ provided excellent accuracy for diagnosing TBM. Conclusions For differentiating TBM from non-TBM individuals, blood and CSF IGRA are good assays and unstimulated CSF IFN-γ is an auxiliary excellent marker.


Search strategy
Our team followed the 2018 guidelines from the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Diagnostic Test Accuracy (PRISMA-DTA) statement published by McInnes et al [22]. This protocol was published on PROSPERO (CRD42021269819). We screened published reports stored in the Web of Science, PubMed, Embase, and the Cochrane Library databases since the inception of English citations indexed up to March 10, 2022. The following search terms were used: (tuberculous meningitis OR tuberculosis meningitis OR tubercular meningitis OR TBM OR meningeal tuberculosis OR extrapulmonary tuberculosis OR EPTB) AND (interferon OR IFN OR interferon-gamma OR gamma-interferon OR interferon-γ OR IFN-gamma OR IFN-γ OR interferon-gamma release assay OR IGRA OR Quanti-FERON-TB OR QFT OR T-SPOT.TB). Furthermore, we reviewed the bibliographies of relevant articles to identify additional potential studies. An agreement of the Institutional Ethics Committee was not required because only publicly available reports were included in the analysis.

Study selection
We selected original studies reporting IFN-γ for diagnosing TBM patients from non-TBM controls using the following criteria: (a) participants including TBM patients and non-TBM controls; (b) index test including two forms of IFN-γ, as TBAg-stimulated IFN-γ (IGRA, including QFT-GIT and T-SPOT.TB; TBAg, including early secretary antigenic target 6 and culture filtrate protein 10) and unstimulated IFN-γ in blood and CSF; (c) diagnostic standard for TBM including microbiologic in CSF (positivity of M. tuberculosis culture or NAAT or polymerase chain reaction, existence of AFB), histopathologic (presence of caseating granuloma in meninges), clinical, radiographic, and laboratory features (adequate resolution of hydrocephalus, granulomas, or basal exudates after empiric antitubercular therapy) [6]; (d) outcomes including sensitivity and specificity of IFN-γ, as well as those where the numerical data computed both these measures; (e) study designs, including randomized controlled trials and prospectively/retrospectively observational trials (cohort and cross-sectional studies). A publication including the largest participant samples was selected from among the identified articles reporting results on overlapping datasets. The optimal cutoff was selected among the articles reporting two or more diagnostic thresholds. The composite definition of the gold standard was adopted, which enrolled definite and probable TBM subjects.
We excluded all studies that were duplicated, non-English and animal publications, editorials and reviews, case reports, abstracts, biochemical and descriptive studies, studies without a control group, and articles presenting original data of less than five participants. Two investigators independently reviewed the titles and abstracts of the citations and full-text of potential studies to ensure that the inclusion criteria were fulfilled. Any disagreements were resolved by consensus.

Data extraction and quality assessment
Two investigators independently extracted the following data from the eligible articles: first author name, date of publication, country where the study was conducted, TB burden, study design, number of participants (TBM/non-TBM individuals), diagnostic reference standard of TBM, and index test including the forms of assay (IGRA or unstimulated IFN-γ), method, cutoff, and TBAg-stimulated condition. In addition, according to different forms of IFN-γ (blood IGRA, CSF IGRA, unstimulated blood IFN-γ, and unstimulated CSF IFN-γ), blood and CSF samples, sensitivity, specificity, true positive (TP), false positive (FP), false negative (FN), and true negative (TN) data were extracted. The Quality Assessment of Diagnostic Accuracy Studies tool-2 (QUADAS-2), recommended by the Cochrane Collaboration, was used to summarize the methodological quality of the selected studies [23]. Deviations were resolved through consensus. The RevMan software (version 5.3; Cochrane, London, UK) was used to generate the figure of quality assessment.

Statistical analysis
A bivariate random model was used to calculate the summary sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) estimates for the different forms of IFN-γ, with 95% confidence intervals (CIs), as we expected significant heterogeneity in eligible studies. Furthermore, the area under the summary receiver operating characteristic curve (AUROC) was calculated. In general, AUROC below 0.75, between 0.75 and 0.93, or exceeding 0.93, indicated that the diagnostic accuracy of IFN-γ was not accurate, good, or excellent, respectively [24]. A hierarchical summary receiver operating characteristic (HSROC) model was constructed to summarize the overall diagnostic accuracy of IFN-γ [25]. We graphically identified the differences in the diagnostic performance across eligible studies using the HSROC curve.
I 2 statistics was used to judge heterogeneity, with I 2 > 0.75 indicating high heterogeneity [26]. The potential causes of heterogeneity were investigated using meta-regression and subgroup analyses. Subgroups were defined as different forms of TBAg-stimulated IFN-γ (T-SPOT.TB and QFT-GIT). Deeks' funnel plot was used to graphically evaluate publication bias, in which P < 0.10 showed substantial asymmetry, suggesting the existence of bias [27]. All data were analyzed using the Stata software (version 14.0; StataCorp, College Station, TX, USA).

Quality of the eligible articles
According to the QUADAS-2 tool, we found a low risk of quality bias for the included articles (Fig 2). Patient selection bias was unclear for five articles (5/20, 25%), as investigators did not report consecutive selection for patient included criteria [33,40,44,45,47]

Diagnostic accuracy of CSF IGRA
Eight studies, comprising 694 CSF samples, were included in the IGRA group. The summary sensitivity and specificity were 0.77 (95% CI: 0.56-0.90; I 2 : 87.76%) and 0.91 (95% CI: 0.85- High heterogeneity was observed between the included studies (I 2 = 87.76% sensitivity). In the meta-regression and subgroup analyses, the different forms of CSF TBAg stimulated IFN-γ (T-SPOT.TB and QFT-GIT) might not be the cause of heterogeneity (P = 0.07). The pooled sensitivity and specificity of CSF T-SPOT.TB were 0.75 and 0.88, respectively. In addition, the pooled sensitivity and specificity of CSF QFT-GIT were 0.81 and 0.98, respectively.

PLOS ONE
Tuberculosis-specific antigen stimulated and unstimulated interferon-γ for tuberculous meningitis diagnosis

Diagnostic accuracy of unstimulated blood and CSF IFN-γ
One study reported a low specificity (0.  5).

Discussion
Our results suggest that blood IGRA demonstrates good diagnostic potential in TBM diagnosis with an AUROC of 0.86. CSF IGRA also showed good diagnostic performance for TBM diagnosis with an AUROC of 0.93. Further, our analysis revealed that the different types of IGRA was not the cause of heterogeneity. In the case of active TB, M. tuberculosis-specific effector T cells could be recruited to the infection site, and then IGRA was used to detect TBAg-stimulated IFN-γ at the infection site (for example, using CSF), which could present a higher specificity of TB diagnosis compared with blood IGRA [20,32,48]. Our study revealed a higher diagnostic specificity (0.91) for CSF IGRA; however, the specificity of blood IGRA was of 0.83.
Our study also showed a slightly higher sensitivity (0.77) of CSF IGRA according to data from eight studies, whereas the sensitivity of blood IGRA was 0.73 based on 15 studies. We assume that the possible reason for lower sensitivity is the protective effect of the blood-brain barrier [32]. Using eight original articles, Yu et al. (2016) performed a meta-analysis of blood and CSF IGRA for distinguishing between TBM and non-TBM individuals [19], in which blood IGRA had a pooled sensitivity of 0.78 and specificity of 0.61, whereas CSF IGRA had a pooled

PLOS ONE
Tuberculosis-specific antigen stimulated and unstimulated interferon-γ for tuberculous meningitis diagnosis

PLOS ONE
Tuberculosis-specific antigen stimulated and unstimulated interferon-γ for tuberculous meningitis diagnosis

PLOS ONE
Tuberculosis-specific antigen stimulated and unstimulated interferon-γ for tuberculous meningitis diagnosis sensitivity of 0.77 and specificity of 0.83. However, this analysis included one article that combined other CNS TB (such as intracranial tuberculoma) patient data into the TBM data [20]. Moreover, three other articles had overlapping datasets [21,48,49]. Therefore, a 2016 new research including the largest participant samples was selected for our study. In addition, the remaining four original articles from the meta-analysis by Yu et al. were selected. We have improved the reliability and stability of the diagnostic performance of IGRA for detecting TBM by analyzing 14 additional publications. Furthermore, we analyzed the unstimulated IFN-γ.
In 2020, Luo et al. included 27 articles and showed that, for TBM diagnosis, the pooled sensitivity and specificity of blood T-SPOT.TB were 0.78 and 0.68, and the pooled sensitivity and specificity of CSF T-SPOT.TB were 0.76 and 0.88 [50]. However, this research also included intracranial tuberculoma patient data for one article [20] and overlapping datasets in four articles [21,36,48,49], causing unreliable pooled results. Besides, 14 eligible articles are in Chinese, which might lead to biased results. Our study included a newest article with the largest participant samples [36], excluding overlapping datasets [21,48,49] and admixture of other brain tuberculosis [20], which conducted more truthful results. Furthermore, we included other 11 original articles and analyzed the performance of QFT-GIT and unstimulated IFN-γ for TBM. In 2021, Lan et al. included 26 publications, and found blood IGRA had a pooled sensitivity of 0.81 and specificity of 0.76 for TBM, and CSF IGRA had a pooled sensitivity of 0.81 and specificity of 0.89 [51]. This meta-analysis had the same problems as Luo et al., in which three included articles had overlapping datasets [21,36,49] and 17 were in Chinese. Our study excluded overlapping datasets [21,49] and Chinese publications, which could increase the reliability of pooled results. Besides, we included other 12 original articles and estimated the detective value of unstimulated IFN-γ.
A systematic review published by Aggarwal et al. in 2021, comprising 7,153 patients, showed that the IFN-γ of unstimulated pleural fluid provided excellent accuracy for diagnosing tuberculous pleural effusion (sensitivity: 0.93, specificity: 0.96) [52]. To date, the diagnostic performance of unstimulated CSF IFN-γ for TBM remained unclear. In our study, unstimulated CSF IFN-γ had a relatively high summary sensitivity (0.86) and specificity (0.92). The AUROC was of 0.95, suggesting that the diagnostic accuracy of unstimulated CSF IFN-γ was excellent. In 2017, Pormohammad et al. performed a systematic review of CSF ADA, indicating that CSF ADA had a relatively high accuracy for TBM diagnosis (sensitivity, 0.89; specificity, 0.91; AUROC, 0.96) [53]. The diagnostic accuracy estimates of the unstimulated CSF IFNγ in the current study are similar to those reported for CSF ADA by Pormohammad et al. Overall, this suggests that unstimulated CSF IFN-γ may be as useful as currently common diagnostic biomarkers, such as ADA, for TBM.
Our meta-analysis had several limitations. First, we could not rule out a misclassification bias because all studies used composite criteria for diagnosing TBM. Second, the different forms of TBAg-stimulated IFN-γ were not the significant source of heterogeneity, however, we did not evaluate other possible factors (such as patient age, HIV-coinfection condition). Furthermore, for TBAg-unstimulated IFN-γ, we did not analyze potential factors for heterogeneity, e.g., clinical types of non-TBM participants, which might influence the interpretability of the pooled results. Third, some studies derived diagnostic thresholds of stimulated IFN-γ in a post-hoc fashion by optimizing the trade-off between specificity and sensitivity, rather than using a manufacturer recommended cutoff, which could be more clinically appropriate for confirmation or exclusion of a TBM diagnosis. Additionally, there was no publication bias in this study; however, we only included studies published in English journals; thus, a concern for publication bias could not be excluded. In clinic, a large CSF sample was needed for the stimulation of IFN-γ among TBM patients with few lymphocytes in the CSF, which is a challenge. Moreover, different age may affect the diagnosis of TBM. In further study, for TBM detection, it is better to explore the pediatric cases and adult patients separately.
In conclusion, this systematic review and meta-analysis shows that blood and CSF IGRA are good assays for differentiating TBM from non-TBM individuals, and unstimulated CSF IFN-γ is an auxiliary excellent marker for detecting TBM. Further large-scale, multi-center, prospective studies are warranted to support our findings, especially the findings on unstimulated CSF IFN-γ.